The ovarian steroid hormones, estradiol and progesterone, regulate functions in the central nervous system resulting in alterations in physiology and reproductive behavior. One means by which estradiol and progesterone exert their neural effects on reproductive behavior is via their interaction with cognate, intracellular receptors functioning as ligand-dependent transcription factors. Studies from my laboratory have shown that in addition to steroid hormones, neurotransmitter dopamine can activate progestin receptors in a ligand-independent manner. Dopamine facilitation of reproductive behavior in female rats and mice occurs by means of cross-talk between membrane receptors for dopamine and intracellular progestin receptors. Signal transduction cascades initiated by dopamine and progesterone, cause an increase in the level of cAMP, PKA activity and phosphorylation of DARPP-32, on Threonine34 in the hypothalamus. In addition to its phosphorylation on Threonine34, DARPP-32 can also be phosphorylated on Threonine75, as well as two Serine residues Ser102 and Ser137. This multisite phosphorylation amplifies the effects of DARPP-32 by converting it into a better substrate for PKA phosphorylation and potentiating its inhibitory effects on the downstream protein phosphatase-1 (PP-1) cascade. While both progesterone and dopamine enhance PKA activity in the hypothalamus, the mechanism(s) by which this increase regulates DARPP-32 inhibition of PP-1 have not been evaluated. This proposal focuses on the mechanisms underlying this multi-site phosphorylation of DARPP-32 by progesterone and dopamine and their effects on female reproductive behavior. Specific aim 1 will evaluate the effects of progesterone and dopamine on DARPP-32 phosphorylation at Thr34 and Thr75 in the hypothalamus and the resultant regulation of PP-1 in mediating the biological effects. Specific aim 2 will determine the modulatory role of dopamine and progesterone on the state of phosphorylation of DARPP-32 on Ser102 and Ser 137 in the hypothalamus and examine their effects on the amplification of DARPP-32/PP-1 cascade. Specific aim 3 will examine the effects of progesterone and dopamine on reproductive behavior using mutant mice with point mutations at Thr34, Thr75, Ser102 and Ser137 of DARPP-32. These studies will provide information on the PKA/DARPP-32/PP-1 cascade regulation by progesterone and dopamine in mediating reproductive behavior.